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IRREGULARITIES IN THE TEST FOR B. COLIIN WATER

RUDOLPH E. THOMPSONReceived for publication, May 25, 1926

The test for B. coli, while considered the most useful methodavailable for determining the sanitary quality of water, is subjectto certain irregularities, chief of which is the occurrence of pre-sumptive positive tests which fail to reveal the presence of B.coli group organisms when subsequently subjected to the usualconfirmatory tests.These non-confirming presumptive positive tests, which occur

with great frequency in certain waters, particularly after chlori-nation, have been ascribed by various investigators to the pres-ence of aerobic and anaerobic spore-forming lactose-fermentingbacteria and to gas-producing symbiotic groups. Severalmediums, notably brilliant green bile, have been experimentedwith for the inhibition of these interfering organisms and exten-sive investigations of a similar nature are at present in progress.

In Toronto, positive presumptive tubes which fail to confirmappear to be of at least two distinct types, (1) those probablydue to one of the above causes, and (2) those in which colon groupbacteria were originally present and lost in the confirmatory pro-cedure. The former, which occur more frequently and constituteover 90 per cent of the presumptive positives from the finallychlorinated water, while consuming a great deal of time and pro-longing the period required for interpretation of results, do notaffect the reliability of the completed tests, whereas the secondtype, in which a negative result is recorded for a tube actuallypositive, may lead to errors of considerable magnitude. It isthis latter type which will be discussed in the present paper.The following examples (table 1), selected from results actuallyobtained in routine examination of raw Lake Ontario water

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when heavy pollution was known to exist, illustrate the extent towhich the value of the B. coli test may be vitiated if the resultsare interpreted on the basis of Standard Methods of WaterAnalysis, i.e., that failure to obtain aerobic colonies on solidmedia inoculated from a positive presumptive tube indicates theabsence of bacteria of the colon group.

It can readily be seen from the above results that the indicatedpollution as judged by the concentration of B. coli would havebeen 100- to 100,000-fold greater if the loss had not occurred.As previously stated these results were obtained when the waterwas undoubtedly polluted to a considerable degree, as indicatedby other tests, chemical and bacteriological, interpreted in the

TABLE 1

B. coli tests on Lake Ontario water-selected resultsThe first, second and third plus or minus in each column indicates the result

of the presumptive test, confirmatory plate and confirmatory broth respectively.EXAMPLE NUMBER 100 cc. 10 cc. 1 cc. 0.1 cc. 0.01 cc. 0.001 cc. 0.0001 cc.

1 +++ +- +- +- ++ +++ -2 ++ +- +- +- +- +- -

5 +++ +- +- +- +++ +++ -6 +- +- +++ +++ +++ _ -7 +++ +- ±- +++ +++ - -8 +++ +- +- +- +++ +++ -

light of past experience with the same source of supply. In thisconnection it is of interest to note the observation of Stearn(1923) that the presumptive test is correct in many cases notproven by the confirmatory tests. Hale (1926) also mentionsthe occurrence of occasional anomalies which indicate that B.coli was originally present but was missed in confirmation.

This type of failure usually occurs when the water is badlypolluted and appears to be seasonal, inasmuch as the majorityof such results are obtained during the period October to April ofeach year. Precott and Winslow (1915) (p. 108, 118), quotingWinslow and Hunnewell (1902), discuss failures with heavilypolluted water which they attribute to overgrowth by sewage

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streptococci and other forms present in sewage water. Anotherexplanation of failures under polluted water conditions whichappears worthy of consideration is that during the growth ofcolon bacteria in standard lactose broth a H-ion concentration isproduced which is lethal to this group of organisms. This firstoccurred to the writer following discussion some few years agowith Mr. Frank Hannan, Chemist, Toronto Filtration PlantLaboratories, in which he advanced the theory that the pollutionof Lake Ontario water during the late summer months would beconsiderably greater if the high pH value obtaining at thatperiod of the year had not an inhibitory effect on the propagationof B. coli, and that the efficacy of lime treatment in destroyingthis group of bacteria was probably due to the pH limit of toler-ance on the alkaline side being exceeded by this process. Scottand McClure (1924) have shown that such is the case. It is thelower limit of tolerance, of course, which is important in regardto the inhibition of B. coli by acid production in carbohydrate-containing media.A survey of the voluminous literature relating to this group of

bacteria provides ample evidence that the life of B. coli in carbo-hydrate media is relatively short and that thepH produced duringthe growth of the organism is the determining factor. It is thisproperty of B. coli that is the basis of the methyl red test of Clarkand Lubs (1915; 1917), the glucose content and buffer value ofthe medium employed being so adjusted that coli cultures elabo-rate acid until the lethalpH zone (rearpH 5) is attained and remainpractically constant, while aerogenes cultures destroy all carbo-hydrate present before reaching this zone and reversion of reac-tion occurs, the pH in the two cases becoming further apart as theperiod of incubation increases. A number of investigators havedetermined and recorded the final pH values of cultures of colongroup organisms, and also the optimum and limiting values forgrowth.

WVhile it appears to be quite generally known that B. coli isinhibited by the acidity formed by its own growth in carbohy-drate media, this fact does not seem to have been seriously con-sidered as a factor in the failure of positive presumptive B. coli

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tests to show the presence of colon bacilli on subsequent examina-tion. That the confirmatory tests should be carried out as soonas possible after fermentation has started in order to avoid lossof B. coli, however, has been observed by several investigatorsand this is a requirement of Standard Methods. Burling andLevine (1918) point out that the confirmatory tests should beperformed as quickly as convenient, preferably in twelve totwenty-four hours, and that an incubation period of forty-eighthours in 1 per cent glucose or lactose is detrimental to the suc-cessful isolation of B. coli; and Hinman (1925), in discussing theuse of inhibitory substances in the preliminary enrichment media,states that the chance of losing weak organisms of the colon groupis much less with lactose broth, especially if the confirmatorytests have been started as soon as gas formation has been demon-strated and before action of the bacteria has produced an unfavor-able acid reaction.

Before describing the experiments carried out brief referencewill be made to the composition of the medium employed, whichwas prepared on the same basis as that used in routine examina-tions in Toronto and in the study of brilliant green bile previouslycarried out (Howard and Thompson, 1925). In the procedurerecommended in Standard Methods, media of the same composi-tion are used in examining both 10 and 1 cc. quantities of water,10 cc. of medium being employed in each case, and as a result thecomposition after dilution with the sample differs considerably.This may not be of importance in regard to the beef extract andpepton content but, as has been shown by Burling and Levine(1918), the lactose content of the mixture of sample and mediumis one of the determining factors in the possibility of loss of colonbacilli during preliminary enrichment. It can readily be seenthat if 10 and 1 cc. respectively are added to two tubes each con-taining 10 cc. of a 0.5 per cent solution of lactose the final lactoseconcentration in the former case will be 0.25 per cent and in thelatter approximately 0.45 per cent. This difference has particu-lar significance in the light of the findings of Chambers (1920)that lethal acidity was produced in 0.4 per cent glucose but thatcontinued growth and reversal of reaction occurred in 0.3 per

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IRREGULARITIES IN TEST FOR B. COLI IN WATER 213

cent glucose after maximum acidity had been reached. It isevident that serious errors might be introduced in this mannerwith a medium containing an inhibitory compound such as bril-liant green. To eliminaite this difficulty, three strengths oflactose broth are employed at Toronto, the composition andamount used being so proportioned that when the desired amountof the water sample has been added the composition of the result-ing mixture will be similar in each case. Three quantities ofwater are employed, namely, 1, 10 and 100 cc., the amount ofmedia per tube being 9, 15 and 50 cc. respectively and the strengthratio 1: 1.5:2.7, taking the strength of standard lactose broth asunity. The final composition in each case under these conditionsis practically the same as when 10 cc. of standard broth is em-ployed with 1 cc. quantities of water according to the procedurerecommended in Standard Methods of Water Analysis.Assuming that the failure of presumptive positive tests to

confirm when the water was known to be polluted was due to theproduction of a lethal H-ion concentration, experiments werecarried out to determine if the death of colon group organismscould be prevented, or at least deferred until the confirmatoryisolation as ordinarily performed had been made, by increasingthe buffer capacity of the medium by the addition of dipotassiumphosphate. The results obtained were most encouraging and indi-cated that the difficulty could be largely eliminated by the em-ployment of an enrichment medium modified in this manner.

All the tests were carried out on 10 cc. quantities of raw LakeOntario water, and the medium employed contained 4.5 gramsbeef extract, 7.5 grams Difco pepton and 7.5 grams lactose perliter. As previously explained, the final composition, afteradding 10 cc. of the water sample to the 15 cc. of medium con-tained in each fermentation tube, was practically the same asthat obtaining when 1 cc. quantities are examined according tothe standard procedure. The desired amount of buffer salt wasadded to this medium just prior to tubing, and tests were thencarried out in parallel with media of the same composition butwithout phosphate. It was observed that a flocculent precipi-tate invariably formed in the buffered medium following sterili-


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RUDOLPH E. THOMPSON

zation in the autoclave. After adding the water, the tubes wereshaken to ensure thorough mixing of the contents. In order toobtain a large number of results in a short period of time fivetubes of each medium were inoculated from each sample of water,the two sets of tubes being subjected to exactly the same con-ditions throughout the test. MacConkey's rebipelagar, de-scribed by Houston (1913) (p. 171), was employed for the con-firmatory tests, this medium having been regularly employed inthe Toronto Laboratories for some years with excellent results.A few preliminary experiments were conducted with lactose

broth of the above strength to which had been added 1 gram ofdipotassium phosphate per liter. The results indicated thatinsufficient buffering capacity was provided by this amount ofphosphate, sterile plates being still obtained, although less fre-quently, and the concentration of buffer salt was therefore in-_creased to 2 grams per liter, corresponding to 1.33 grams perliter of standard strength broth. The results with the latterconcentration are shown in tables 2, 3 and 4. Series 1 was carriedout on consecutive working days during the spring of 1925,series 2 on several periods of consecutive days during the fall of1925 and the spring of 1926 and series 3 on selected days in thespring of 1926 when pollution was known to be present. Theconditions existing on the days on which the tests comprisingseries 3 were carried out were such that all positive presumptivetests could be attributed to the presence of colon group organisms,thus eliminating interference from positive tests due to otherundefined causes when the water is relatively unpolluted, whichcannot readily be distinguished in summarized data from thosedue to the death of colon bacteria during the enrichment process.A summary of all results is given in table 5.These results indicate that a considerable loss is apt to occur

with preliminary enrichment in lactose broth and that addition ofdipotassium phosphate to the medium aids in the eli ination ofthis source of error. As previously stated the occurrence of thesefailures is seasonal, and the frequency of occurrence varieswidely. During the period when series 1 was carried out the losswas particularly marked, while in series 2 it was hardly apparent.

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It should be pointed out that the three negative confirmatoryplates from the buffered medium in Series 2 were obtained whenthe water was relatively unpolluted and were probably due tocauses other than production of a lethal H-ion concentration. Inseries 3, carried out on selected days when pollution was knownto be present, the loss is again evident but not as marked as inseries 1. The two sterile plates obtained with thespecialmedium

TABLE 2

Series I

STANDARD

STANDARD BROTH CON-STANDARD TAININGBROTH 0.2 PZR CENT

KHPO4

Number of tubes inoculated.......................... 100 100

Presumptive:Positive 24 hours................................. 74 77Positive 48 hours................................. 6 9Negative 48 hours................................ 20 14

Confirmatory plates:24-hour presumptives: Positive................... 45 77

Negative.................. 29 048-hour presumptives: Positive ........ ........... 1 3

Negative.................. 5 6

Confirmatory broths:24-hour presumptives: Positive................... 45 77

Negative.................. 0 048-hour presumptives: Positive................... 1 2

Negative.................. 0 1

Total number positiveconfimd46 79

in this series were undoubtedly due to the production of lethalacidity notwithstanding the presence of the buffer salt. Theorganisms present on the days on which these two failures oc-curred were apparently particularly active in the production ofacid as on both occasions the other four plates in the group werevery thinly populated and of the five parallel plates from thestandard medium only one on each occasion was positive, a soli-tary colony developing in each instance.


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RUDOLPH E. THOMPSON

Simply recording the number of positive and negative resultsdoes not adequately describe the difference in the plates preparedfrom the two media. In the majority of instances a much largernumber of colonies was obtained from the buffered medium thanfrom the standard broth inoculated from the same sample ofwater, and when the number from the former was low a portion ofthe latter were usually sterile. When the water was polluted gas

TABLE 3

Series 2

STANDARD

TAAD BROTH CON-SRTAHAR TAlININBROTHN 0.2 PER CENT

K2HPO,

Number of tubes inoculated........................... 100 100

Presumptives:Positive 24 hours................................. 51 53Positive 48 hours................................. 9 14Negative 48 hours................................ 40 33

Confirmatory plates:24-hour presumptives: Positive ................... 46 50


Negative.................. 6 8

Confirmatory broths:24-hour presumptives: Positive................... 46 50


Negative.................. 0 0

Total number positive confirmed.49 56

production in the presumptive tubes was usually greater and lessvariable in the buffered medium, undoubtedly due to the exten-sion of the period of activity of the organism. In this connectionit is of interest to note the observation of Bronfenbrenner andSchlesinger (1918) that the amount of gas produced during thefermentation of lactose by B. coli varies inversely with the H-ionconcentration, other factors being equal, or directly with the

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concentration of buffer. Besson, Ranque and Senez (1919) alsodiscuss the relationship of gas production and acid production byB. coli in glucose-containing media, which they believe to betwo distinct processes occurring practically coincidently.The confirmatory plates were prepared by diluting one loop-

ful of the presumptive culture with 7 cc. of sterile distilled waterand adding one loopful of the resulting mixture to a tube of melted

TABLE 4

Series 3

STANDARDBROTH

STANDARDBROTH CON-TAINING

0.2 PFR CENTK2HP04

Number of tubes inoculated........................... 100 100

Presumptive: Positive 24 hours....................... 100 100

Confirmatory plates: Positive........................ 74 98Negative....................... 26 2

Confirmatory broths: Positive........................ 71 97Negative....................... 3 1

Total number positiveconfirmed.71 97

TABLE 5Summary of all results

STANDARD

STANDARD BROTH CON-SRTANAR TAININGBROTH 0.2 PFR CIENT

KsHPO4

Number of tubes inoculated.......................... 300 300Total number positive confirmed..................... 166 232

rebipelagar, which was then mixed and poured into a sterile Petridish. This procedure, with practice, yields well-distributedplate cultures from normal presumptive positive tubes, but careis required to avoid overcrowding. A sterile plate obtainedunder these conditions does not necessarily indicate the absenceof colon group organisms in the presumptive tube, but only thatthe concentration of bacteria is insufficient to give a positive

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result at this great dilution. In previous work, presumptivetubes which had given a sterile plate by this method were foundby the writer to give well-distributed positive plates when aloopful of the presumptive medium was transferred directly tothe agar medium, omitting the dilution with sterile water. Toproduce such a result there must be a deviation from the numberof viable organisms per unit volume normally present in the pre-sumptive medium of the order of 10,000-fold. To overcome sucha condition either two suspensions of these widely varying dilu-tions, and possibly others to avoid sterility on one hand and over-crowding and coalescence on the other, would have to be platedor the presumptive tubes would have to be retained for furtherexamination in event of failure of the first test. Both of theseprocedures would introduce complications and add to the volumeof work, and would, of course, be useless if death of the culturehad occurred. There appears to be little doubt that modifica-tion of the presumptive medium to defer the rapid destruction ofcolon group organisms by the acidity produced offers the mostsatisfactory solution of the problem.Another irregularity, described in a previous paper (Howard

and Thompson, 1925) is evident in the results of series 3, i.e.,failure of typical colonies to produce gas when fished into lactosebroth as the final step in the confirmatory procedure. This, atfirst, was also attributed to the production of lethal acidity, butthe theory was later disproven as it was found that a typicallypositive confirmatory plate could be obtained by subculturingthe negative confirmatory broth in the usual manner. Failuresof this type are not confined to the MacConkey medium, assimilar results were obtained when employing eosine methyleneblue agar, and the cultures formed typical colonies on Endomedium also. For the purpose of the present study these fail-ures can be disregarded as their occurrence appears to be inde-pendent of the preliminary enrichment medium employed.

Other modifications of the preliminary enrichment mediumwhich seemed worthy of consideration were increasing the buffercapacity of the medium by employing a larger percentage ofpepton (Mfiller 1922 and Steam 1923) and decreasing the

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anmount of acid produced by further reducing the concentrationof lactose. Brief series of comparative experiments were carriedout with standard broth modified in each of these particulars.The former proved quite ineffective, lactose broth containingtwice (1 per cent) and four times (2 per cent) the prescribed con-centration of pepton giving approximately the same number offailures as the standard medium. With regard to the employ-ment of broth containing less than the standard amount (0.5per cent) of lactose, sufficient experiments have not been carriedout to warrant any definite conclusion. A brief series of testsconsisting of fifty tubes of standard broth inoculated in parallelwith fifty tubes of broth containing one-fifth (0.1 per cent) of thestandard concentration of lactose showed that while the failureswere eliminated the broth of low lactose content was less sensitivewith relatively unpolluted water, and the total number of positivetests confirmed was therefore practically the same. There wasalso a tendency for the confirmatory plates made from the brothcontaining 0.1 per cent lactose to be overgrown with alkali-forming organisms. This appears to indicate that the acidformed in the presumptive medium aids in the elimination ofother forms present in the water under examination and that itserves a useful function in inhibiting the growth of organismswhich would otherwise interfere with the isolation of colon groupbacteria. If such is the case it would be undesirable to reduce thelactose content to such an extent that inhibition of these otherbacteria would be prevented. Further experiments with inter-mediate concentrations of lactose and with other buffering agentsshould be undertaken. It is possible that a medium embracing acombination of these modifications might provide optimum con-ditions for the enrichment of B. coli. Studies along these lines,however, will have to be postponed until the fall of the year,when the failures will probably again become prevalent.The writer wishes to emphasize the fact that modification of

the standard medium to increase its buffering capacity is notsuggested as a means of eliminating spurious positive tests due tothe presence of aerobic and anaerobic lactose-fermenting spore-formers, symbiotic complexes, etc., and that the prevention of


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the type of failure herein discussed would by no means eliminatethe desirability of developing a medium which would be toxic toorganisms giving rise to false presumptive tests and yet besatisfactory for the enrichment of colon group bacteria. Thecomparative tests conducted with brilliant green bile indicatedthat the possibility of loss of typical organisms during the con-firmatory procedure was very much less in this medium than instandard lactose broth, probably due to the reduced activity ofthe bacteria in the presence of the inhibitory compound. Theemployment of such a medium would probably incidentallyeliminate failures due to the production of lethal acidity.

In conclusion the writer wishes gratefully to acknowledge thekindly criticism and advice of Mr. Norman J. Howard, Bacteriol-ogist in Charge of the laboratories in which this work wascarried out, and Mr. Frank Hannan, Chemist. The mediaemployed in the investigation were prepared by Mr. Chas. Lepperof the laboratory staff.

SUMMARY

1. Failures of presumptive positive B. coli tests of pollutedwater to show the presence of colon group organisms on subse-quent examination are recorded which are believed to be due tothe production of a lethal H-ion concentration during prelimin-ary enrichment.

2. It is pointed out that while it is generally known that B.coli is inhibited by the acidity formed by its own growth incarbohydrate-containing media, this fact does not seem to havebeen seriously considered as a factor in the failure of presumptivetests to confirm.

3. Results of comparative tests are given which indicate thatfailures due to the production of a lethal H-ion concentrationmay be largely eliminated by increasing the buffering capacity ofthe presumptive medium by the addition of -dipotassiumphosphate.

REFERENCESBESSON, A., RANQUE, A., AND SENEZ, CH. 1919 Compt. rend. soc. biol., 82, 78-8,BRONFENBRENNER, J., AND SCHLESINGER, M. J. 1918 Proc. Soc. Exp. Biol. Med.,

16, 44 46.

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JRREGULARITIES IN TEST FOR B. COLI IN WATER 221

BURLING, H. A., AND LEVINE, MAX. 1918 Amer. Jour. Pub. Health, 8, 306-307.CHAMBERS, W. H. 1920 Ann. Missouri Bot. Gardens, 7,249-289.CLARK, W. M., AND LulBS, H. A. 1915 Jour. Inf. Dis., 17,160.CLARK, W. M., AND Lu-BS, H. A. 1917 Jour. Biol. Chem., 30,209-234.HALE, F. E. 1926 Amer. Jour. Pub. Health, 16, 428-431.HINMAN, J. J., JR. 1925 Amer. Jour. Pub. Health, 15, 614-619.HOUSTON, A. C. 1913 Studies in Water Supply.HOWARD, N. J., AND THOMPSON, R. E. 1925 Can. Eng., 48, 413-417.MtLLER, F. 1922 Biochem. Z., 131,485-498.PRESCOTT, S. C., AND WINSLOW, C.-E. A. 1915 Elements of Water Bacteriology.SCOTT, R. D., AND MCCLURE, G. M. 1924 Jour. Amer. Water Works Assoc.,

11, 598-604.STEARN, E. W. 1923 Amer. Jour. Pub. Health, 13,567-70.WINSLOW, C.-E. A., AND HUNNEWELL, Mf. P. 1902 Jour. Med. Res., 8,502.


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